What is CRISPR-Cas9 in Gene Editing?

What is CRISPR-Cas9?

The CRISPR-Cas system is a natural immune system in prokaryotes. Some bacteria, upon being invaded by viruses, can store a small segment of the viral genes in a region within their DNA known as CRISPR. Upon encountering the virus again, the bacteria can recognize the virus based on the stored segment, enabling them to cleave the viral DNA, rendering it ineffective. The invading foreign DNA is cleaved by Cas nucleases and is then captured and integrated into the CRISPR locus as spacer sequences flanked by conserved repeat sequences. These acquired spacer sequences serve as templates for creating short CRISPR RNAs (crRNAs). Together with trans-activating crRNA, they form a complex that acts as a guide strand, directing the Cas9 nuclease to the complementary invading DNA. Once bound, the Cas9 protein cleaves the crRNA-complementary and antisense strands through its HNH and RuvC-like nuclease domains, respectively.

Figure 1. Mechanisms of CRISPR-Cas-based genome editing or modification.

Components of CRISPER-Cas9 System

The CRISPR-Cas9 genome editing system consists of two main components, including the endonuclease Cas9 and the guide RNA (gRNA).

(1) The endonuclease is the Cas9 nuclease protein derived from Streptococcus pyogenes. Cas9 nuclease possesses active domains for cleaving double-stranded DNA, which includes the RuvC-like and HNH-like nuclease domains, leading to the breakage of the double-stranded DNA.

(2) The gRNA is a chimeric RNA formed by combining a scaffold function provided by tracrRNA with a specific crRNA. Later, these two RNA segments are fused into a single short RNA, forming the sgRNA.

(3) The 5' end of the gRNA consists of a 20-bp sequence that is specific for binding, serving as the target-recognition mechanism. It guides the Cas9/gRNA complex to the specific DNA target through RNA-DNA base pairing.

Adjacent to the specific binding sequence is the Protospacer Adjacent Motif (PAM) sequence. The PAM sequence is the recognition site for the Cas9 endonuclease, and for Cas9, the PAM sequence is 5'-NGG. The Cas9 nuclease cleaves the double-stranded DNA at the third base upstream of the PAM sequence.

How CRISPR-Cas9 Works?

Stage 1: Acquisition of CRISPR's highly variable spacer region (Capture of foreign DNA)

In this stage, the highly variable spacer region of CRISPR is acquired, which means that a small DNA sequence from an invading phage or plasmid DNA is integrated into the host bacterium's genome, located between the two repeat sequences at the 5' end of the CRISPR locus. This records the chronological order of invasion by foreign genetic material in the CRISPR locus. The acquisition of new spacer sequences typically involves three steps:

(1) Proteins encoded by Cas1 and Cas2 scan the invading DNA, identify the PAM region, and select an adjacent DNA sequence as a candidate prototype spacer.

(2) The Cas1/2 protein complex cuts and excises the prototype spacer sequence from the foreign DNA and, with the assistance of other enzymes, inserts it downstream of the CRISPR sequence's leader region.

(3) DNA undergoes repair to seal the open double-stranded gap, adding the new spacer sequence to the genome's CRISPR sequence.

Stage 2: Expression of the CRISPR locus (including transcription and post-transcriptional maturation)

The CRISPR sequence is transcribed under the regulation of the leader region, producing pre-crRNA (the precursor of crRNA). Simultaneously, tracrRNA (trans-activating crRNA) with a complementary sequence to the pre-crRNA is also transcribed. The pre-crRNA forms a double-stranded RNA with tracrRNA through complementary base pairing and assembles with the protein encoded by Cas9 to create a complex. This complex selects the appropriate "identification number" (spacer sequence RNA) based on the invader's type and, with the assistance of Ribonuclease III (RNase III), trims this "identification" into a short crRNA, consisting of a single type of spacer sequence RNA and part of the repeat sequence region.

Stage 3: Activation of the CRISPR/Cas system (Precision interference)

The end-stage complex comprises crRNA, Cas9, and tracrRNA, functioning like a guided missile to precisely strike at the invader's DNA. This complex scans the entire foreign DNA sequence, identifying the original spacer sequence complementary to crRNA. At this point, the complex localizes to the region of the PAM/original spacer sequence, opening the DNA double strand to form an R-loop. One strand of the DNA hybridizes with crRNA, while the other remains free.

Subsequently, the Cas9 protein makes a precise cut in the DNA double strand three nucleotides upstream of PAM, generating a blunt-end cut. The HNH domain of the Cas9 protein is responsible for cutting the DNA strand that complements crRNA, while the RuvC domain cuts the other non-complementary DNA strand. Finally, under the action of Cas9, a double-strand break (DSB) occurs in the DNA, leading to silencing the expression of foreign DNA, and the invader is eliminated.

Advantages of CRISPR-Cas9

• Simple to operate with high precision in targeting.
• Capable of achieving knockout at individual target gene loci or multiple loci simultaneously.
• Can be used for simultaneous knockout of multiple target genes.
• Applicable to any species.
• The CRISPR-Cas9 system is RNA-guided and can lead to heritable modifications in genes.

Differences between CRISPR-Cas9 and RNAi

What is CRISPR-Cas9 in Gene Editing?

- mg g kg ton

We recommend you to leave us your work email address so that we can serve you better. Please leave your organization information here if you don't have a work email address.

SUBMIT